1. Field of the Invention
The present invention relates to a novel method for testing vascular endothelial damage and a testing kit.
2. Description of Related Art
Metabolic syndrome is a generic term used to refer to a pathological condition which is a complication of visceral fat obesity and any two or more of hyperglycemia, hypertension and hyperlipemia. This metabolic syndrome is a pathological condition which has to be prevented or improved from the viewpoint of reducing medical cost and securing QOL of aged people for a coming aging society. As for the metabolic syndrome, it has been considered effective to prevent from progress of the symptom by improving a lifestyle.
For the progress of pathogenesis of the metabolic syndrome, it has been acknowledged that the vascular endothelial damage is involved. For example, it has been reported that a population of patients who received damage in the vascular endothelium has a higher incidence of cardiovascular event.
By the way, it is known that activated various cells release vesicle called microparticle (MP; MicroParticle). These microparticles with maximum diameter to 1 or to 2 μm are known as platelet-derived microparticle (PDMP), endothelial cell-derived microparticle (EDMP), monocyte-derived microparticle (MDMP), and the like, depending on derived cell, and are understood that they are playing important role in the living organism.
These MPs contain various kinds of membrane proteins (GpIIb/IIIa, VE-cadherin, and Tissue Factor (TF)), and the like, and are considered to have a role in thrombus formation. In addition, a high MP level in blood circulation has been observed in various pathological conditions. Therefore, it is anticipated that detection and quantitative determination of these MPs may be an important criteria in detection and evaluation of severity of precursor state of thrombosis in the progress of various pathological conditions, and thus, in the evaluation of pathological condition and severity of the metabolic syndrome. And, if preventive medical care based on these evaluations can be realized, usefulness thereof in an aging society which will come from now on is immeasurable.
Conventionally, as to testing techniques of various pathological conditions relevant to the vascular endothelial damage using microparticle as a marker of pathological conditions, for example, JP-A-2003-533698 describes a method for detecting and monitoring the precursor state of thrombosis using platelet-derived microparticle (PDMP) as a marker. In addition, JP-A-2008-529702 describes a method for diagnosing cardiovascular disease, evaluation of prognosis thereof, or evaluation of the presence of progress thereof, similarly using the platelet-derived microparticle (PDMP) as a marker.
Conventionally, as to a method for detection and quantitative determination of the microparticle in a biological sample (for example, in the plasma), for example, a filtration method, an enzyme-linked immunosorbent assay (ELISA) method, and the like have been proposed. However, in the recent years, detection and quantitative determination of MP are widely performed through the use of the flow cytometry method by which size and surface antigen marker of MP can be measured simultaneously. Principle of the flow cytometry method, and the advantage of the method that can identify and determine quantitatively a partial population of cells and cell particles are nowadays well known to the person skilled in the art. And also, in the application other than detection and quantitative determination, the flow cytometry method has already been used, for example, for detection of activated platelet in the blood, and detection of platelet-derived prohemostatic microparticle, and so on. In addition, using the flow cytometry method, multiple measurement objects can be measured simultaneously. Despite this, the method for detection and quantitative determination of MP by the flow cytometry method which has been proposed conventionally is a method for measuring only one type of MP using monochromic fluorescent marker. In this regard, conventional technology has not sufficiently taken advantages of the flow cytometry method.